PROJECT SUMMARY/ABSTRACT This project is entitled ?MicroRNAs as Predictors of Crohn's Disease Phenotype and Regulators of Intestinal Epithelial Cell Biology?. There is an increasing appreciation for the importance of variable cellular processes in intestinal epithelial cells (IECs) that contribute to the loss of barrier capacity and the development of Crohn?s disease (CD). We and others have shown that alterations in IEC gene expression are associated with CD, but the underlying mechanisms remain unresolved. Our multi-disciplinary team provides critical complementary expertise in high-throughput genomics (RNA-seq, small RNA-seq, and single cell RNA-seq), intestinal epithelial cell biology, and cutting-edge technologies for functional validation (patient crypt-derived colonoids). MicroRNAs (miRNAs) are small non-coding RNAs that confer post-transcriptional regulation of gene expression. Using next- generation sequencing, we recently identified that miRNAs in colon tissue, in particular miR-31, stratify CD patients into subtypes that are associated with different clinical progressions. We demonstrated that miR-31-5p is upregulated most dramatically in IECs isolated from patients with CD. Cellular mechanisms affected by miR- 31-5p activity in IECs are unknown. We showed that upregulated miR-31-5p results in decreased expression of colonocyte and goblet cell marker genes implying altered differentiation, which could in turn mitigate barrier function. We hypothesize that miR-31-5p contributes to CD phenotypes through regulation of epithelial specific genes that are critical for proper IEC differentiation and maintenance of the intestinal barrier. In Aim 1, in order to establish the utility of miRNAs for predicting CD disease behavior, we will conduct small RNA-seq on RNA isolated from index colon and ileal biopsies (obtained at time of diagnosis) in formalin-fixed, paraffin-embedded tissue from a large cohort of treatment-nave and well-phenotyped pediatric patients with CD and non-IBD controls (with a minimum 5-year longitudinal follow-up at the University of North Carolina Hospitals). In Aim 2, we will determine the role of miR-31-5p in IEC behavior, differentiation, and defects in barrier function using gain- and loss-of-function experiments for miR-31-5p in human crypt-derived colonoids from CD patients and non-IBD controls. We will also conduct complementary studies in mice over-expressing miR-31-5p. In Aim 3, we will test whether miR-31-5p modulates the expression of genes in IECs that contribute to altered IEC differentiation. In particular, we will test whether ALK1, a component of the type 1 receptor for TGF-beta signaling molecules, which we have identified as a target of miR-31-5p in human colon, is involved in the regulation of IEC differentiation in CD via control of the NOTCH signaling pathway. To define comprehensively the targets for miR- 31-5p (as well as all for other miRNAs) in IECs, we will also perform AGO-CLIP followed by sequencing (AGO- CLIP-seq) using isolated IECs and primary cultured IECs from CD patients and non-IBD controls. Our proposal is significant because it will establish the prognostic utility and functional relevance of specific miRNAs in CD. This study will lay the groundwork for improved clinical trial designs and novel therapeutic modalities.